Preparation method and application of Lactobacillus rhamnosus LRa66 postbiotics for improving rhinitis

Abstract

The present invention discloses a preparation method and application of a postbiotic of Lactobacillus rhamnosus LRa66 for improving rhinitis, comprising inoculating Lactobacillus rhamnosus LRa66 on an MRS medium for culturing and then crushing under high pressure. The postbiotic prepared by the present invention has the function of alleviating cell barrier damage and inflammatory response caused by excessive histamine secretion, which is specifically embodied in: improving the transmembrane resistance of HNEpC cell monolayers; downregulating the permeability of HNEpC cell monolayers; inhibiting excessive secretion of mucin MUC5AC; restoring the abnormal expression of barrier proteins KLK5 and KLK7; and reducing the transcriptional expression of inflammatory pathway PI3Kα / IκBα / IKKβ.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing, and specifically relates to a preparation method and application of a Lactobacillus rhamnosus LRa66 postbiotic for improving rhinitis. Background Art

[0002] Rhinitis is an inflammation of the nasal mucosa and is a broad term that covers inflammatory diseases of the nasal mucosa caused by a variety of different factors. These factors include infection, allergic reactions, environmental irritants, mechanical stimulation, drug reactions, etc. Allergic rhinitis (AR), also known as allergic rhinitis, is a specific type of rhinitis caused by an abnormal response of the immune system to allergens in the environment (such as pollen, dust mites, pet dander, mold spores, etc.). This reaction usually involves the production of immunoglobulin E (IgE), an antibody in the body that can recognize and bind to specific allergens, triggering a series of inflammatory responses.

[0003] When pollen, dust mites, etc. are inhaled, they bind to specific IgE antibodies that have been adsorbed on the surface of mast cells in the nasal mucosa, triggering these cells to release inflammatory mediators such as histamine. These inflammatory mediators stimulate the sensory nerve endings and blood vessels of the nasal mucosa, excite the parasympathetic nerves, and trigger an immediate phase reaction, leading to symptoms such as nasal itching, sneezing, and clear watery nasal discharge. Subsequently, histamine and other inflammatory mediators induce vascular endothelial cells, epithelial cells, etc. to express or secrete adhesion molecules, chemokines, and cytokines, etc., recruit and activate immune cells, resulting in persistent and aggravated inflammatory reactions, and obvious tissue edema of the nasal mucosa, leading to nasal congestion.

[0004] Currently, the commonly used drugs for the treatment of allergic rhinitis include cetirizine, loratadine, montelukast, etc. These drugs can relieve symptoms, but usually cannot correct the underlying immune disorder, so they cannot completely cure rhinitis, and may have side effects, such as long-term or improper use of drugs may cause side effects, such as nasal glucocorticoids may cause nasal mucosal atrophy, antihistamines may cause drowsiness or dry mouth, etc.

[0005] Postbiotics are a class of preparations of inanimate microorganisms and / or their components that are beneficial to the health of the host. Some studies have shown that specific postbiotics may have an ameliorative effect on AR. Studies have shown that postbiotics can strengthen the intestinal epithelial barrier by enhancing the tight junctions of epithelial cells and promoting the expression of tight junction proteins such as claudin and occluding, thereby restoring and maintaining the integrity of the intestinal barrier and reducing the invasion of pathogens and harmful substances.

[0006] However, the postbiotic preparations used to improve rhinitis in the existing technology are still very limited, and the effect of these preparations in improving rhinitis still needs to be further improved.

[0007] Therefore, how to provide a postbiotic preparation that can effectively improve and treat rhinitis and will not cause adverse reactions in patients during the treatment process has become an urgent problem to be solved by those skilled in the art. Summary of the invention

[0008] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0009] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0010] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing a Lactobacillus rhamnosus LRa66 postbiotic.

[0011] In order to solve the above technical problems, the present invention provides the following technical solution: a method for preparing a postbiotic of Lactobacillus rhamnosus LRa66, characterized by comprising inoculating Lactobacillus rhamnosus LRa66 on MRS culture medium for culturing and then crushing it under high pressure.

[0012] As a preferred embodiment of the preparation method of the present invention, the high-pressure crushing conditions are: pressure 250-310 MPa, temperature 4-10° C., and homogenization 8-10 times.

[0013] Another object of the present invention is to overcome the deficiencies in the prior art and provide a Lactobacillus rhamnosus LRa66 postbiotic for improving rhinitis, characterized in that the Lactobacillus rhamnosus LRa66 postbiotic comprises Lactobacillus rhamnosus LRa66.

[0014] As a preferred embodiment of the Lactobacillus rhamnosus LRa66 postbiotic of the present invention, the Lactobacillus rhamnosus LRa66 strain is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with the deposit number being CGMCC No.24282.

[0015] Another object of the present invention is to overcome the deficiencies in the prior art and provide an application of a Lactobacillus rhamnosus LRa66 postbiotic for improving rhinitis, characterized in that the Lactobacillus rhamnosus LRa66 postbiotic can improve and repair nasal mucosal barrier damage, nasal mucosal barrier dysfunction and inflammatory damage caused by excessive histamine secretion.

[0016] As a preferred embodiment of the application of the present invention, the Lactobacillus rhamnosus LRa66 postbiotic can enhance the transmembrane resistance of HNEpC cell monolayer.

[0017] As a preferred embodiment of the application of the present invention, the Lactobacillus rhamnosus LRa66 postbiotic can down-regulate the permeability of HNEpC cell monolayer.

[0018] As a preferred embodiment of the application of the present invention, the Lactobacillus rhamnosus LRa66 postbiotic can inhibit excessive secretion of mucin MUC5AC.

[0019] As a preferred embodiment of the application of the present invention, the Lactobacillus rhamnosus LRa66 postbiotic can restore the abnormal expression of barrier proteins KLK5 and KLK7.

[0020] As a preferred embodiment of the application of the present invention, the Lactobacillus rhamnosus LRa66 postbiotic can reduce the transcriptional expression level of the inflammatory pathway PI3Kα / IκBα / IKKβ.

[0021] Beneficial effects of the present invention:

[0022] The postbiotics prepared by the present invention have the effects of alleviating cell barrier damage and inflammatory response caused by excessive histamine secretion, which are specifically embodied in: improving the transmembrane resistance of HNEpC cell monolayers; downregulating the permeability of HNEpC cell monolayers; inhibiting excessive secretion of mucin MUC5AC; restoring the abnormal expression of barrier proteins KLK5 and KLK7; and reducing the transcriptional expression of inflammatory pathway PI3Kα / IκBα / IKKβ. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0024] Figure 1 This is a graph showing the effect of Lactobacillus rhamnosus LRa66 on the transepithelial electrical resistance of the cell monolayer barrier.

[0025] Figure 2 This is a graph showing the effect of Lactobacillus rhamnosus LRa66 on cell barrier permeability.

[0026] Figure 3 This is a graph showing the effect of Lactobacillus rhamnosus LRa66 on the secretion level of cellular MUC5AC.

[0027] Figure 4 This is a result diagram of the effect of Lactobacillus rhamnosus LRa66 on the transcriptional expression of cell barrier proteins of the present invention, wherein: Figure 4 (A) is KLK5; Figure 4 (B) is KLK7.

[0028] Figure 5 This is a result diagram of the effect of Lactobacillus hamnosus LRa66 on the transcriptional expression of cellular inflammatory pathways, wherein: Figure 5 (A) is PI3Kα; Figure 5 (B) is IκBα; Figure 5 (C) is IKKβ. DETAILED DESCRIPTION

[0029] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0030] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0031] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0032] Unless otherwise specified, all raw materials used in the examples of the present invention are commercially available, as shown in Table 1 for details.

[0033] Table 1

[0034]

[0035] The present invention provides a rhamnosus lactobacillus LRa66 for improving rhinitis, and the rhamnosus lactobacillus LRa66 for improving rhinitis is named as the rhamnosus lactobacillus LRa66 strain, the deposit unit is the General Microbiological Center of the China Microbiological Culture Collection Administration, the deposit number is CGMCC No.24282, the deposit date is January 10, 2022, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. It was first disclosed in Chinese patent CN202310481728.X.

[0036] The human nasal mucosal epithelial cells (HNEpC) used in the experiment were purchased from Dico (Shanghai) Biotechnology Co., Ltd.

[0037] MRS solid medium: weigh 10 g of peptone, 10 g of beef extract, 20 g of glucose, 2 g of sodium acetate, 5 g of yeast powder, 2 g of diammonium hydrogen citrate, 2.6 g of K2PO4·3H2O, 0.1 g of MgSO4·7H2O, 0.05 g of MnSO4, 20 g of agar, and 1 g of L-cysteine, dissolve them in deionized water, add 1 mL of Tween 80, make up to 1 L, sterilize and cool, then pour into a sterilized culture dish for later use.

[0038] MRS liquid culture medium: weigh 10 g of peptone, 10 g of beef extract, 20 g of glucose, 2 g of sodium acetate, 5 g of yeast powder, 2 g of diammonium hydrogen citrate, 2.6 g of K2PO4·3H2O, 0.1 g of MgSO4·7H2O, 0.05 g of MnSO4, and 1 g of L-cysteine, dissolve them in deionized water, add 1 mL of Tween 80, make up to 1 L, sterilize and cool for later use.

[0039] Example 1

[0040] Isolation and screening of Lactobacillus rhamnosus LRa66:

[0041] Breast milk samples were selected and diluted 10-fold using 0.9% saline solution for 3 times. The samples were spread on MRS solid culture medium and cultured at 37°C for 48 hours. Single colonies were picked and streaked on the surface of MRS solid culture medium for purification. Single colonies were picked again and expanded using MRS liquid culture medium at 37°C and preserved using 30% glycerol.

[0042] Example 2

[0043] Identification of Lactobacillus rhamnosus LRa66:

[0044] (1) Morphological identification:

[0045] The strain was inoculated into MRS solid medium, cultured at 37°C for 48 h, and then observed under a microscope.

[0046] Observation shows that the colonies are milky white, semicircular in shape, with a smooth and moist surface and neat edges.

[0047] (2) 16S rRNA molecular biological identification:

[0048] The strain stored at -80°C was taken out, inoculated into a centrifuge tube containing 20 mL of MRS liquid culture medium at a ratio of 2%, cultured at 37°C for 12-18 hours, and then centrifuged at 8000 rpm for 10 minutes, the supernatant was removed, and the bacteria were collected.

[0049] The genome of the strain was extracted, and bacterial universal primers were added for PCR amplification. The amplified products were sent to Suzhou Genewise Biotechnology Co., Ltd. for sequencing and identification.

[0050] The strain was sequenced and analyzed, and its 16S rDNA sequence was shown as SEQ ID No.1.

[0051] The sequenced sequences were compared with the nucleic acid sequences in the National Center for Biotechnology Information, and the results showed that the strain was indeed Lactobacillus rhamnosus.

[0052] Example 3

[0053] Preparation of postbiotic samples of Lactobacillus rhamnosus LRa66:

[0054] Lactobacillus rhamnosus LRa66 was inoculated into MRS liquid culture medium, cultured at 37°C for 12-18h, centrifuged at 8000rpm for 10min, the supernatant was removed, the bacteria were collected, the bacterial concentration was adjusted to 109CFU / mL with PBS, high-pressure homogenized at 300MPa pressure, 4-10°C for 8-10 times, centrifuged at 8000rpm for 10min, the supernatant was filtered with a 0.22μm filter membrane, and stored at -20°C.

[0055] Example 4

[0056] The repair effect of Lactobacillus rhamnosus LRa66 postbiotic sample on the damage of nasal mucosal epithelial cell monolayer barrier:

[0057] The frozen HNEpC cells were quickly thawed at 37°C, and the cells were resuspended in three volumes of cell culture medium. The cells were centrifuged at 500 g for 5 min, the supernatant was discarded, the cells were resuspended in cell culture medium again, and inoculated into a T25 cell culture dish.

[0058] The revived cells were cultured under 5% CO2 and 37°C. When about 80%-90% of the area of ​​the cell culture dish was covered by growing cells, the cells were subcultured. Digested with 1 mL of trypsin-EDTA (0.25%), observed under a microscope, and when the cells were suspended, digestion was immediately terminated with 3 volumes of complete cell culture medium. After centrifugation to collect the cells, they were subcultured at a ratio of 1:2. The experimental group settings are shown in Table 2, in which Yin ginseng group 2 was the intervention of postbiotic samples of Lactobacillus rhamnosus WKA55.

[0059] Table 2 Grouping information table

[0060]

[0061]

[0062] (1) Effects of Lactobacillus rhamnosus LRa66 postbiotic samples on cell barrier integrity

[0063] After three generations, cells in the logarithmic phase were cultured at 2×10 4 The cells were inoculated in the Transwell plate at a density of 10 cells / well. Before establishing the monolayer barrier model, the upper chamber of the Transwell was moistened with pre-cooled PBS solution in advance, and then the cells were inoculated in the upper chamber of the Transwell plate. After the cells grew continuously for 15 days, a continuous and dense cell monolayer can be formed in the upper chamber of the Transwell plate, and subsequent experimental treatments can be performed at this time. The entire experiment was divided into three groups: a blank group, a HIS modeling group, and a postbiotic treatment group. The experiment used a segmented incubation model, in which the blank group represented the addition of only complete culture medium without any experimental treatment; the modeling group represented the addition of HIS to the complete culture medium, and after incubation with cells for 24 hours, PBS was used to wash it off; the postbiotic treatment group represented the addition of HIS to the cells after 24 hours of incubation with PBS, and at this time, a 40-fold diluted postbiotic sample was added and incubated with cells for 6 hours. The TEER value was measured using a resistor before and after treatment. The change in TEER value was calculated as follows: TEER / (Ω·cm 2 )=(TEER cell group-TEER blank group)×Transwell polycarbonate membrane area, TEER(%)=TEER after intervention / TEER before intervention×100.

[0064] Transepithelial electrical resistance (TEER) is a commonly used method to assess the integrity of cell barriers, especially in in vitro models. TEER measures the electrical resistance across the cell layer, which reflects the tightness of the cell-cell junctions, i.e. the integrity of the barrier. The higher the TEER value, the stronger the barrier function of the cell layer and the greater the resistance to the passage of substances. The test results of the TEER value are expressed as the rate of change of the TEER value, such as Figure 1 As shown. Compared with the blank group, histamine significantly induced a 79.53% decrease in the transepithelial resistance of the HNEpC cell monolayer (P<0.0001), indicating that the integrity of the cell barrier was destroyed. After LRa66 intervention, compared with the model group, the transepithelial resistance of the cell monolayer increased by 76.90% (P<0.0001), and the loratadine group was 48.54%, WKA55 was 41.81%, and PBS had no significant effect. Their effects on increasing the transepithelial resistance of the cell monolayer were inferior to LRa66, indicating that LRa66 has the most significant effect on improving the integrity of the cell barrier in the histamine-induced HNEpC cell inflammation model.

[0065] (2) Effects of Lactobacillus rhamnosus LRa66 postbiotic samples on cell barrier permeability

[0066] After equilibration in a 37°C incubator with phenol red-free DMEM (without serum and double antibody) for 2 hours, the cells were washed with PBS three times, and the upper chamber of the Transwell was replaced with 1 mg / mL FITC-Dextran prepared in phenol red-free DMEM, while the lower chamber was still phenol red-free DMEM. After 1 hour, 200 μL of the lower chamber was transferred to a 96-well black bottom plate and detected with an ELISA reader, with an excitation wavelength of 495 nm and an emission wavelength of 525 nm. The permeability change of monolayer cells to FITC-Dextran is expressed by the permeability coefficient Papp, which is calculated as follows: Papp = (dQ / dt) / (A×C0), Papp (%) = Papp before intervention / Papp after intervention × 100. Where: Papp is the permeability coefficient (cm / s), dQ / dt is the amount of substance passing through the cell monolayer per unit time (μg / s), A is the surface area of ​​the cell monolayer (cm2), and C0 is the initial concentration of the substance in the upper chamber (μg / mL).

[0067] A healthy cell barrier has appropriate permeability, which can maintain the stability of the body's internal environment and protect the body from harmful external substances. When the cell barrier is damaged or its function declines, its permeability will increase, causing substances that should not pass through to enter the cell layer, which may cause pathological conditions such as inflammatory response, tissue damage or infection. The test results of cell barrier permeability are expressed as the rate of change of permeability coefficient, such as Figure 2 As shown. Compared with the blank group, histamine significantly induced an increase in the permeability of the HNEpC cell monolayer by 72.33% (P<0.05), which indicates that the cell barrier function was damaged to a certain extent. After LRa66 intervention, the cell monolayer permeability decreased by 41.33% (P<0.05) compared with the model group, and the loratadine group, WKA55 and PBS intervention had no significant effect on the cell monolayer permeability. This shows that LRa66 has the most significant effect on the repair of the cell barrier function in the histamine-induced HNEpC cell inflammation model.

[0068] Example 5

[0069] Improvement effect of Lactobacillus rhamnosus LRa66 postbiotic sample on abnormal barrier function of nasal mucosal epithelial cells:

[0070] The frozen HNEpC cells were quickly thawed at 37°C, and the cells were resuspended in three volumes of cell culture medium. The cells were centrifuged at 500 g for 5 min, the supernatant was discarded, the cells were resuspended in cell culture medium again, and inoculated into a T25 cell culture dish.

[0071] The revived cells were cultured under 5% CO2 and 37°C. When about 80%-90% of the area of ​​the cell culture dish was covered by growing cells, the cells were subcultured. Digested with 1 mL of trypsin-EDTA (0.25%), observed under a microscope, and when the cells were suspended, digestion was immediately terminated with 3 volumes of complete cell culture medium. After the cells were collected by centrifugation, they were subcultured at a ratio of 1:2.

[0072] After three passages, the cell concentration was adjusted to 2 × 10 5 / mL, 100μL of cell suspension was inoculated in each well of a 96-well plate, and 1.5mL of cell suspension was inoculated in each well of a 12-well plate. After the cells adhered to the wall, the experimental groups were set up as shown in Table 1. Except for the blank group, the cells were intervened with 200μM histamine overnight, washed with PBS three times, and the cells were intervened with different samples for 3h.

[0073] (1) Effects of postbiotic samples of Lactobacillus rhamnosus LRa66 on cell mucin secretion levels

[0074] After 1 hour of excitation, the 12-well plate was discarded of the supernatant, washed three times with PBS, and 400 μL of 0.5% TritonX-100 was added to each well to completely dissolve the cells. After 30 minutes, the plate was centrifuged at 2000 rpm for 10 minutes, and the supernatant was taken for the detection of MUC5AC secretion level. The standard was diluted and added to the enzyme-labeled plate, and standard solutions of different concentrations were prepared, and then the samples to be tested were added and diluted. After the plate was sealed, it was incubated at 37°C for 30 minutes, and then the plate was washed 5 times with diluted washing solution. After that, the enzyme-labeled reagent (except the blank well) was added, and it was incubated and washed again. When developing color, color developer A was added first, and then color developer B was added, and the color was developed in the dark for 15 minutes. After terminating the reaction, the absorbance of each well at a wavelength of 450nm was measured, and the entire determination process must be completed within 15 minutes after terminating the reaction.

[0075] MUC5AC is an important mucin in respiratory mucus. Its abnormal expression is associated with a variety of airway mucus hypersecretion diseases, including asthma, chronic obstructive pulmonary disease, chronic sinusitis, etc. In rhinitis, especially allergic rhinitis, increased expression of MUC5AC is a characteristic change. Figure 3As shown in the figure, compared with the blank group, the MUC5AC secretion level in the model group increased by 40.77% (P<0.0001), reaching 69.33μg / L. After LRa66 intervention, compared with the model group, the MUC5AC secretion level decreased by 36.25% (P<0.0001), and the loratadine group decreased by 11.18% (P<0.01). WKA55 and PBS intervention had no significant effect on the cell MUC5AC secretion level. This shows that LRa66 can significantly alleviate the problem of aggravated inflammation caused by abnormal MUC5AC secretion in HNEpC cells induced by histamine.

[0076] (2) Effects of postbiotic samples of Lactobacillus rhamnosus LRa66 on the expression of tight junction proteins in cells

[0077] After 1 hour of excitation, 500 μL Trizol was added to the 12-well plate to extract total RNA, and the OD260 / OD280 values ​​were detected. After reverse transcription was completed according to the instructions of the reverse transcription kit, the cDNA sample was diluted 20 times and the fluorescence real-time quantitative reaction was performed according to the instructions of the q-PCR kit. The primers are shown in Table 3.

[0078] Table 3 Primer sequences

[0079]

[0080] KLK protein (kallikrein-related peptidase) plays an important role in maintaining cell barrier function, especially in the physiological and pathological processes of the skin barrier. Abnormal expression or increased activity of KLK protein is associated with a variety of epithelial tissue lesions. Its overactivation can lead to keratinocyte proliferation and differentiation, cell barrier function damage and inflammation. The results of the detection of KLK transcriptional expression are as follows Figure 4 As shown, compared with the blank group, the model group KLK5 ( Figure 4 A) and KLK7( Figure 4 B) The expression at the transcriptional level was significantly increased (P<0.0001).

[0081] After LRa66 intervention, the expression of KLK5 transcription level was affected as follows Figure 4 As shown in A, compared with the model group, KLK5 expression was downregulated by 63.84% (P<0.0001), and the loratadine group decreased by 24.88% (P<0.0001) after intervention, and WKA55 decreased by 44.69% (P<0.0001). PBS intervention had no significant effect on cells. Figure 4As shown in B, compared with the model group, after LRa66 intervention, KLK7 expression was downregulated by 41.21% (P<0.0001), and loratadine, WKA55 and PBS intervention had no significant effect on the cells. This shows that LRa66 can significantly improve the abnormal expression of barrier proteins in HNEpC cells induced by histamine, help enhance cell barrier function and relieve inflammation.

[0082] (3) Effects of Lactobacillus rhamnosus LRa66 postbiotic samples on the expression of cellular inflammatory pathways

[0083] After 1 hour of excitation, 500 μL Trizol was added to the 12-well plate to extract total RNA, and the OD260 / OD280 values ​​were detected. After reverse transcription was completed according to the instructions of the reverse transcription kit, the cDNA sample was diluted 20 times and the fluorescence real-time quantitative reaction was performed according to the instructions of the q-PCR kit. The primers are shown in Table 4.

[0084] Table 4 Primer sequences

[0085]

[0086] PI3Kα, IκBα, and IKKβ are key molecules in the inflammatory response. PI3Kα participates in the inflammatory response by activating the Akt signaling pathway, regulating cell growth and survival, and affecting the activation of NF-κB. IκBα, as an inhibitory protein of NF-κB, maintains the inactive state of NF-κB in the cytoplasm; when stimulated by inflammation, IKKβ activates the phosphorylation of IκBα, leading to its degradation, releasing NF-κB into the nucleus and activating the expression of inflammatory genes. IKKβ also regulates the homeostasis of inflammation by competitively phosphorylating AMPK and IκBα. Therefore, the interaction and activity changes of these molecules jointly determine the intensity and persistence of the inflammatory response, and are potential targets for anti-inflammatory treatment.

[0087] The results of the detection of PI3Kα transcriptional expression in cells are as follows Figure 5 As shown in A, compared with the blank group, the expression of PI3Kα in the model group increased by 4.25 times (P<0.0001). After LRa66 intervention, PI3Kα expression decreased by 68.59% (P<0.0001), and was downregulated by 17.25% (P<0.01) after loratadine intervention, 50.53% (P<0.0001) after WKA55 intervention, and 15.89% (P<0.01) after PBS intervention. This shows that LRa66 has the most significant effect on inhibiting the expression of PI3Kα transcription level.

[0088] The results of the detection of the expression of IκBα transcription level in cells are as follows Figure 5As shown in B, compared with the blank group, the expression of IκBα in the model group increased by 2.02 times (P<0.0001). After LRa66 intervention, the expression of IκBα decreased by 39.64% (P<0.0001), and was downregulated by 17.29% (P<0.01) after loratadine intervention, downregulated by 28.61% (P<0.0001) after WKA55 intervention, and had no significant effect on the cells after PBS intervention. This shows that LRa66 has the most significant effect on inhibiting the expression of IκBα transcription level.

[0089] The results of the detection of cell IKKβ transcriptional expression are as follows Figure 5 As shown in C, compared with the blank group, the expression of IKKβ in the model group increased by 1.75 times (P<0.0001). After LRa66 intervention, IKKβ expression decreased by 43.65% (P<0.0001), and loratadine intervention downregulated it by 28.46% (P<0.01). WKA55 and PBS intervention had no significant effect on the cells. This shows that LRa66 has the most significant effect on inhibiting the expression of IKKβ transcription level.

[0090] In summary, LRa66 can significantly inhibit the expression of the inflammatory pathway PI3Kα / IκBα / IKKβ, effectively reduce the inflammatory response, and provide a potential therapeutic strategy for the treatment of rhinitis.

[0091] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

[0092] The 16S rDNA sequence is:

[0093]

Claims

1. A method for preparing a postbiotic of Lactobacillus rhamnosus LRa66, characterized in that: The method comprises the following steps: inoculating Lactobacillus rhamnosus LRa66 on MRS medium for cultivation and then crushing the culture medium under high pressure.

2. The preparation method according to claim 1, characterized in that: The high-pressure crushing conditions are: pressure 250-310 MPa, temperature 4-10° C., and homogenization 8-10 times.

3. A Lactobacillus rhamnosus LRa66 postbiotic for improving rhinitis, characterized in that: The Lactobacillus rhamnosus LRa66 postbiotics include Lactobacillus rhamnosus LRa66.

4. The Lactobacillus rhamnosus LRa66 postbiotic according to claim 3, characterized in that: The rhamnosus Lactobacillus LRa66 strain is deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration, with the deposit number CGMCC No. 24282 and the deposit date January 10, 2022.

5. An application of Lactobacillus rhamnosus LRa66 postbiotics for improving rhinitis, characterized in that: The Lactobacillus rhamnosus LRa66 postbiotic can improve and repair nasal mucosal barrier damage, nasal mucosal barrier dysfunction and inflammatory damage caused by excessive histamine secretion.

6. The use according to claim 5, characterized in that: The Lactobacillus rhamnosus LRa66 postbiotic can enhance the transmembrane resistance of the HNEpC cell monolayer.

7. The use according to claim 6, characterized in that: The Lactobacillus rhamnosus LRa66 postbiotic can downregulate the permeability of HNEpC cell monolayers.

8. The use according to claim 7, characterized in that: The Lactobacillus rhamnosus LRa66 postbiotic can inhibit the excessive secretion of mucin MUC5AC.

9. The use according to claim 8, characterized in that: The Lactobacillus rhamnosus LRa66 postbiotic can restore the abnormal expression of barrier proteins KLK5 and KLK7.

10. The use according to claim 9, characterized in that: The Lactobacillus rhamnosus LRa66 postbiotic can reduce the transcriptional expression level of the inflammatory pathway PI3Kα / IκBα / IKKβ.

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